The apple FERONIA receptor‐like kinase MdMRLK2 negatively regulates Valsa canker resistance by suppressing defence responses and hypersensitive reaction

Abstract Valsa canker, caused by the fungus Valsa mali, is one of the most destructive diseases of apple trees in China and other East Asian countries. The plant receptor‐like kinase FERONIA is involved in plant cell growth, development, and immunity. However, little is known about the function of FERONIA in apple defence against V. mali. In this study, we found that MdMRLK2 was highly induced by V. mali in twigs of V. mali‐susceptible Malus mellana but not in those of the resistant species Malus yunnaensis. 35S:MdMRLK2 apple plants showed compromised resistance relative to wild‐type (WT) plants. Further analyses indicated that 35S:MdMRLK2 apple plants had enhanced abscisic acid (ABA) levels and reduced salicylic acid (SA) levels relative to the WT on V. mali infection. MdMRLK2 overexpression also suppressed polyphenol accumulation and inhibited the activities of phenylalanine ammonia‐lyase (PAL), β‐1,3‐glucanase (GLU), and chitinase (CHT) during V. mali infection. Moreover, MdMRLK2 interacted with MdHIR1, a hypersensitive‐induced response protein, and suppressed the MdHIR1‐mediated hypersensitive reaction (HR), probably by impairing MdHIR1 self‐interaction. Collectively, these findings demonstrate that overexpression of MdMRLK2 compromises Valsa canker resistance, probably by (a) altering ABA and SA levels, (b) suppressing polyphenol accumulation, (c) inhibiting PAL, GLU, and CHT activities, and (d) blocking MdHIR1‐mediated HR by disrupting MdHIR1 self‐interaction.


| INTRODUC TI ON
Apple (Malus domestica) is a popular temperate fruit that has long been appreciated for its unique characteristics and rich nutrition (Daccache et al., 2020;Sun et al., 2020). China is the largest producer of apples worldwide . Apple trees are highly vulnerable to many diseases, including those caused by fungi, such as Marssonina blotch (Diplocarpon mali) (Zhao et al., 2013), powdery mildew (Podosphaera leucotricha) (Tian et al., 2019), bitter rot (Colletotrichum acutatum) (Jurick et al., 2011), and Valsa canker (Valsa mali) (Wang et al., 2011a(Wang et al., , 2011b. Valsa canker caused by V. mali occurs with an annual average incidence of approximately 52.7% (Meng et al., 2019). Valsa canker is widespread and destructive: it causes the death of twigs, limbs, and finally the whole tree, reducing apple production and causing significant economic losses in China (Song et al., 2020). However, the molecular defence mechanisms of apple plants against V. mali infection are poorly understood, and only a few effective management strategies have been reported to date.
Plant resistance to pathogens is a dynamic and complex biological process that involves various changes at the biochemical, molecular, and physiological levels (AbuQamar et al., 2017). When a plant detects an attempted pathogen invasion, it rapidly activates sophisticated defence mechanisms to protect itself from foreign threats. The activation of complex phytohormone signalling networks is vital, as it stimulates the plant immune signalling network (Robert-Seilaniantz et al., 2011;Yang et al., 2015). Salicylic acid (SA) is an important plant hormone that plays a critical role in plant disease resistance either by promoting the synthesis of preformed or inducible antimicrobial defence compounds termed phytoalexins or by activating defence signalling (Kumar, 2014;Vlot et al., 2009). SA is required for the activation of systemic acquired resistance, which is marked by increased expression of many defence proteins, including pathogenesis-related (PR) proteins (Kumar, 2014). Systemic acquired resistance is a form of systemic immunity that protects distal, uninfected parts of the plant against secondary infections by related or unrelated pathogens (Kachroo et al., 2020). Plants deficient in SA signalling are incapable of developing systemic acquired resistance and do not show pathogenesis-related gene activation on pathogen infection (Pieterse et al., 2009). Li et al. (2019a) demonstrated that exogenous SA improved tomato resistance to tomato yellow leaf curl virus. Application of exogenous SA has also been reported to induce resistance to Glomerella leaf spot in apple cv. Gala leaves (Zhang et al., 2016). In addition to SA, other plant hormones such as jasmonic acid (JA) and abscisic acid (ABA) also trigger and modulate plant resistance to biotrophic and necrotrophic pathogens through a complex signalling network. JA plays an essential role in plant defence responses against pathogens, especially fungi . Mutants of JA biosynthesis and signalling genes display increased susceptibility to various fungi, and studies have shown that SA acts antagonistically to JA (Mur et al., 2006). Whether or not the JA signalling pathway is enhanced also depends on the lifestyle of the pathogen. ABA has been shown to enhance susceptibility in other plant-pathogen systems (Adie et al., 2007). For example, early studies showed that pretreatment of potato plants with ABA increased their susceptibility to Phytophthora infestans and Cladosporium cucumerinum (Henfling et al., 1980). In wheat, Puccinia striiformis f. sp. tritici stimulates ABA accumulation, also promoting fungal infection (Huai et al., 2019).
Phenolic compounds are important plant secondary metabolites whose production by the shikimate-phenylpropanoid pathways is enhanced under stress conditions (Rasouli et al., 2016). Phenolic compounds are important for the induction of plant resistance (Mandal et al., 2010). For example, the synthesis of phenolic compounds is triggered in cells adjacent to injured tissues to restrict pathogen spread from local sites (Ferreira et al., 2007). In particular, phenolic acids, the main components of phenolic compounds, are ubiquitous in plants and can be incorporated into the cell wall in response to biotic stress (Oliveira et al., 2020;Zafari et al., 2016). Some plants respond to pathogen attack by accumulating phenolic acids such as gallic, ferulic, p-coumaric, and chlorogenic acids. Studies have shown that some phenolic acids are frequently involved in the plant defence system; one example is p-coumaric acid, which was reported to be positively correlated with fungal incidence (Giorni et al., 2020). In addition to phenolic acids, defence-related enzymes including phenylalanine ammonia-lyase (PAL), β-1,3-glucanase (GLU), and chitinase (CHT) are also induced to defend against Fusarium sulphureum in potato (Yu et al., 2016). Additional studies have shown that PAL, GLU, and CHT activities increase significantly in the presence of Pseudoperonospora cubensis and enhance the resistance of cucumber leaves (Shi et al., 2007). Tian et al. (2006) reported that elicitors significantly enhanced defence-related enzyme activities to defend against Alternaria rot in pear.
Phytopathogenic microorganisms are common in nature and pose a constant threat to plants. Nonetheless, plants rarely become infected and develop disease; a multilayered innate immune system protects them from most pathogens (Johansson et al., 2015). To cope with pathogens, infected plants may deploy a rapid and strong defensive response called the hypersensitive reaction (HR) (Balint-Kurti, 2019). The HR is a local cell death response at the site of infection that involves highly dynamic reorganization of host cells and often manifests as localized programmed cell death (PCD), which effectively prevents the spread of biotrophic pathogens (Balint-Kurti, 2019;Higaki et al., 2011;Liu et al., 2005). An HR typically occurs during successful defence in host plants, usually leaving only small necrotic spots . Wang et al. (2009) observed that Arabidopsis thaliana with delayed HR showed compromised resistance to Pseudomonas syringae pv. tomato (Pto) DC3000. Studies also showed that HR inhibition allowed Phaerotheca fuliginea to penetrate and form haustoria in wheat . Although significant research efforts have focused on the regulation of plant HR, many questions about potential mechanisms remain to be addressed. Plants employ multiple mechanisms to suppress the inappropriate activation of HR and to constrain it after activation because of its potentially severe costs (Balint-Kurti, 2019). Previous studies have demonstrated that HIR1 associates with the plasma membrane and triggers hypersensitive cell death in rice and pepper (Choi et al., 2011;Zhou et al., 2010).
Plants have evolved an intricate system to control HIR1-mediated HR, and among the negative regulators of this response are the so-called leucine-rich repeat (LRR) proteins (Jung & Hwang, 2007).
Small LRR proteins have been reported to negatively modulate HIR1-mediated HR during pathogen attack (Choi et al., 2011). LRR domains exist in most receptor-like kinases (RLKs) and participate in signal transduction for disease resistance (Hosseini et al., 2020).
Plant RLKs and receptor-like proteins can rapidly recognize invading pathogens . Recently, many researchers have reported that the RLK FERONIA is involved in plant responses to pathogen invasion (Liao et al., 2017). For example, fer mutants have been shown to be less susceptible to the powdery mildew Golovinomyces orontii . Pathogenic fungi produce RALF (Rapid Alkalinization Factor) 1-like peptide to activate FER signalling events, including apoplastic alkalinization, that in turn activate Fmk1 in the fungus to enhance virulence (Masachis et al., 2016).
FERONIA, a receptor for the RALF peptide ligand, integrates a number of regulatory pathways that target cell expansion, energy metabolism, immune responses, and abiotic stress responses (Liao et al., 2017;Stegmann et al., 2017). Previous studies have shown that some pathogenic fungi produce RALF-like peptides to activate the host FERONIA-mediated pathway and thus increase their virulence and cause plant disease (Liao et al., 2017). Although many studies suggest that FERONIA is involved in immune responses in a complex with other proteins (Masachis et al., 2016;Xiao et al., 2019), no direct evidence has yet been provided on the role of FERONIA in apple defence against V. mali infection. By MdMRLK2 expression levels that were increased 15.2-and 19-fold, respectively (Figure 1d,e). The protein level of MdMRLK2 in wildtype (WT), OE-1, and OE-2 apple plants was analysed, which clearly showed that the two OE lines expressed full-length MdMRLK2 and the MdMRLK2 bands were stronger in OE lines than in WT plants ( Figure 1f). In addition, we inoculated leaves and twigs of WT and OE lines with V. mali. Three days after inoculation, the lesion areas were clearly larger in the OE lines than in WT plants (Figure 1g,h).
By 5 days postinoculation (dpi), the twig lesion lengths were significantly longer in OE lines than in WT plants (Figure 1i,j). We also inoculated three MdMRLK2 RNAi apple calli lines with V. mali. The lesion areas were significantly larger in WT calli than in the MdMRLK2 RNAi lines, and the WT calli showed more cell death than the MdMRLK2 RNAi lines based on trypan blue staining ( Figure S1). These results indicated that MdMRLK2 plays a negative role in V. mali resistance.

| Overexpression of MdMRLK2 increased ABA but reduced SA content of apple plants on V. mali infection
We next measured the contents of three hormones with important roles in disease resistance: ABA, SA, and JA. There were no differences in ABA content between WT and OE lines at day 0, but at 3 dpi the ABA level was 50.2% higher in leaves of 35S:MdMRLK2 lines than in leaves of WT plants ( Figure 2a). In twigs, the level of ABA was 3.6-fold and 3.3-fold higher in OE-1 and OE-2 plants, respectively, compared with the WT (Figure 2b). By contrast, the leaf SA content was 18.8% and 26.9% lower in OE-1 and OE-2 than in WT plants (Figure 2c), and the SA content in twigs showed a similar trend ( Figure 2d). Plant resistance-related genes such as PR1, PR4, PR5, and PAL were expressed at higher levels in leaves and twigs of WT plants than in those of OE lines ( Figure S2). There was no significant difference in JA content between 35S:MdMRLK2 lines and WT plants ( Figure S3). To verify the effects of ABA and SA in V. mali resistance, ABA and SA were sprayed on leaves before inoculation with V. mali; the lesion areas were clearly larger following ABA treatment and smaller following SA treatment compared with those of the controls (Figure 2e).

| Overexpression of MdMRLK2 inhibited PAL, GLU, and CHT activities during V. mali infection in apple plants
Further analyses were performed to determine whether the activities of disease-related enzymes increased after V. mali inoculation.

| MdMRLK2 interacted with MdHIR1 and limited the HR mediated by MdHIR1
To explore the molecular mechanism by which MdMRLK2 compromised V. mali resistance, we performed yeast two-hybrid (Y2H) screening and found that MdMRLK2 targeted hypersensitive-induced

| DISCUSS ION
Valsa canker, a destructive disease of apple trees, is caused by the ascomycete V. mali (Lee et al., 2006;Li et al., 2013;Wang et al., 2014). The pathogen typically invades apple trees through wounds or natural ostioles in the bark, and it induces severe tissue maceration and necrosis (Feng et al., 2021). Valsa canker was first identified in Japan and is now widespread in eastern Asia, where it causes severe yield losses each year and has a profound effect on apple production . However, the molecular mechanisms that underlie apple response to V. mali infection remain unclear.
FERONIA acts as a sensor of cell wall integrity during the hostpathogen interaction and triggers further downstream immune responses in the host cell . The immune responses triggered by FERONIA in response to fungal and bacterial pathogens were initially reported by Keinath and Kessler and colleagues Kessler et al., 2010). Previous studies have shown that two rice FERONIA-like receptor genes, OsFLR2 and OsFLR11, attenuate the resistance of rice seedlings to Magnaporthe grisea by downregulating defence-related genes and suppressing ROS bursts . In Arabidopsis, FERONIA mutations confer increased resistance to G. orontii and Fusarium oxysporum, but reduce resistance to Hyaloperonospora arabidopsidis and Colletotrichum higginsianum Masachis et al., 2016). FERONIA has diverse functions in response to different pathogens based on their trophic type. In this study, Hormones are key regulators of plant growth, development, and defences (Franck et al., 2018), and multiple hormone signalling pathways are integrated to boost plant immunity in response to biotic stresses. In Arabidopsis, FERONIA is involved in the crosstalk between several hormone pathways that regulate cell growth, seed yield, and stress responses (Franck et al., 2018;Liao et al., 2017). After plants were infected with V. mali in the present study, leaf ABA levels were 50.2% higher in the 35S:MdMRLK2 lines than in the WT (Figure 2a), and twig ABA levels increased drastically in 35S:MdMRLK2 lines compared with the WT (Figure 2b). By contrast, SA levels were lower in 35S:MdMRLK2 than in the WT (Figure 2c,d).
We also treated WT leaves with exogenous ABA and SA before V. mali inoculation, and the lesion areas were larger after ABA treatment and smaller after SA treatment compared with the control signalling, negatively influencing plant immunity (Guo et al., 2018).
In this study, there was no difference in JA content between 35S:MdMRLK2 and WT plants ( Figure S2).
Phenolic acids have been frequently described as contributing to defence against plant fungal pathogens, either through direct interference with the fungus or through reinforcement of plant structural components that act as a mechanical barrier (Gauthier et al., 2016;Lattanzio et al., 2006;Siranidou et al., 2002). In response to pathogen infection, phenolic acids are released from the cell wall or massively synthesized by the plant, accumulating rapidly at the infection sites (Atanasova-Penichon et al., 2012). Among the phenolic acids, derivatives of cinnamic acid (e.g., caffeic, ferulic, and p-coumaric acids) are the best recognized contributors to fusarium head blight resistance (Gauthier et al., 2015). Here, we measured phenolic acids in apple leaves and twigs, and found that the contents of phenolic acids (gallic acid, ferulic acid, p-coumaric acid, and chlorogenic acid) were significantly higher in WT plants than in 35S:MdMRLK2 plants ( Figure 3). This result demonstrates that MdMRLK2 overexpression is detrimental to polyphenol accumulation. Host protection against fungal pathogen invasion is due in large part to a defence system that is highly coordinated to prevent the spread of pathogens Yu et al., 2016). PAL is a key enzyme in the phenylpropanoid pathway, which is responsible for aspects of the host defence system (Huang et al., 2010). CHT degrades chitin, which is the major component of fungal pathogen cell walls. GLU, one of the most fully characterized pathogenesis-related proteins, also acts indirectly by releasing an oligosaccharide and eliciting defence reactions, then acting synergistically with CHT to inhibit fungal growth (Ji et al., 2021). BiFC and split-luciferase complementation assays confirmed this interaction (Figure 5b,c). A co-immunoprecipitation assay also indicated that MdMRLK2 interacted with MdHIR1 ( Figure 5d). Induction of HIR genes occurs in response to attacks by various pathogens, including bacteria, fungi, and viruses, and the accumulation of HIR proteins induces host cell death and disease resistance (Duan et al., 2013;Jung & Hwang, 2007;Li et al., 2019b;Qi et al., 2011). The HR is defined as rapid cell death that occurs in the region of invasion; it limits pathogen spread, prepares the plant defence system for successive assaults, and is closely related to active resistance (Choi & Hwang, 2015;Noman et al., 2020;Pontier et al., 1998). Because MdMRLK2 interacts with MdHIR1, we hypothesized that this in-

| Materials and treatments
GL-3, isolated from cv. Royal Gala, was used for apple transformation. The transformation method was similar to that described by Dai et al. (2013). Tissue-cultured WT and transgenic apple plants were subcultured every 4 weeks. The rooting method for WT and transgenic plants was based on that described in Sun et al. (2018).
The rooted WT and transgenic apple plantlets were cultivated on rooting medium for 40 days and then transferred to pots (8 × 8 cm) that contained nutrient soil, vermiculite, and perlite mixed in a 3:1:1 ratio. Inoculation was performed as described by Suzaki et al. (1997) with the minor modifications mentioned by Feng et al. (2020). Prior to inoculation, fully expanded apple leaves were surface-disinfected with 0.6% sodium hypochlorite solution and rinsed three times by spraying with sterile water. V. mali strain 03-8 was cultured on potato dextrose agar for 3 days. Agar plugs (5 mm each) were taken from the margin of the growing colony and placed on the abaxial leaf surface by the needle-stab method. Twigs were cut into 20-cm segments and washed with tap water, immersed in 0.6% sodium hypochlorite for 6 min, and rinsed with sterile water three times. The ends of the twigs were sealed with wax. Each twig segment was subjected to wounding with a hole puncher (Xu et al., 2018). Agar plugs without fungus were used as negative controls. The twigs and leaves were placed horizontally in a plastic box, which was immediately cov-

| Y2H and Y3H assays
A partial coding sequence for the MdMRLK2 protein (469-892 amino acids) was cloned into the pGBKT7 vector as bait for Y2H screening.
The coding sequence of MdHIR1 was inserted into the pGADT7 and pGBKT7 vectors (primers are listed in Table S1). The Y2H assay was performed as described by Petzold et al. (2018

| BiFC assay
The coding sequences of MdMRLK2 and MdHIR1 were inserted into the 35S:pSPYCE-cYFP vector, and the coding sequence of MdHIR1 was inserted into the 35S:pSPYNE-nYFP vector. We used an apple aquaporin protein MdPIP2, a plasma membrane protein, as negative control. Leaves from 5-week-old N. benthamiana were used for the BiFC assay, and fluorescence was detected as described by Wang et al. (2020). Confocal imaging was performed using an FV3000 confocal laser scanning microscope (Olympus). The primers used for vector construction are listed in Table S1.

| Split-luciferase complementation assay
The coding sequence of MdHIR1 without the stop codon was inserted into pCAMBIA1300-nLUC, and the coding sequences of MdMRLK2 and MdHIR1 were cloned into pCAMBIA1300-cLUC.
The split-luciferase complementation assay was performed by transient expression in leaves of N. benthamiana by agroinfiltration as described by Fernandez et al. (2020). Leaves that co-expressed different constructs were examined for luciferase activity by applying 1 mM d-luciferin and placing them in the dark for 5 min before imaging. Luciferase complementation was observed with a CCD imaging system (Lumazone Pylon 2048B) using 10-min exposures.

| Statistical analysis
The data were analysed using one-way analysis of variance followed by Tukey's multiple comparison test (p < 0.05) in SPSS 20.0 (IBM). Innovation from Shaanxi Province (2020zdzx03-01-01).

CO N FLI C T O F I NTE R E S T
The authors declare that they have no conflict of interest.

DATA AVA I L A B I L I T Y S TAT E M E N T
The data supporting the findings of this study are available from the corresponding author upon reasonable request.